We describe a highly disabling congenital myasthenic syndrome (CMS) associated with rapidly decaying, low-amplitude synaptic currents, and trace its cause to a valine to leucine mutation in the signature cystine loop (cys-loop) of the AChR α subunit. The recently solved crystal structure of an ACh-binding protein places the cys-loop at the junction between the extracellular ligand-binding and transmembrane domains where it may couple agonist binding to channel gating. We therefore analyzed the kinetics of ACh-induced single-channel currents to identify elementary steps in the receptor activation mechanism altered by the αV132L mutation. The analysis reveals that αV132L markedly impairs ACh binding to receptors in the resting closed state, decreasing binding affinity for the second binding step 30-fold, but attenuates gating efficiency only about twofold. By contrast, mutation of the equivalent valine residue in the δ subunit impairs channel gating approximately fourfold with little effect on ACh binding, while corresponding mutations in the β and ε subunits are without effect. The unique functional contribution of the α subunit cys-loop likely owes to its direct connection via a β strand to αW149 at the center of the ligand-binding domain. The overall findings reveal functional asymmetry between cys-loops of the different AChR subunits in contributing to ACh binding and channel gating.
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